Altered response to Wnt signaling drives abnormal stem cell states. (A) Receptor–ligand interaction analyses from scRNAseq data of control (Ctr) and CerS4^epi−/− skin. Communication within OB cells is illustrated. Note altered Wnt signaling in CerS4^epi−/− OB. (B) KEGG- and GOBP-term enrichment analyses of differentially expressed genes in CerS4^epi−/− OB stem cells. (C) Experimental outline for noncanonical Wnt inhibition using 5 μM KN93 or 5 μM SP600125. (D) FACS-based quantification of HFSCs from Ctr and CerS4^epi−/− organoids treated with KN93 or SP600125. Note an increase in HFSCs in KN93-treated CerS4^epi−/− organoids and a decrease in HFSCs in SP600125-treated CerS4^epi−/− organoids (n = 4 mice/genotype, mean ± SD; unpaired t test). (E) Representative images and quantification of Ctr and CerS4^epi−/− organoids treated with KN93 and stained for keratin-6 (grey) and keratin-14 (magenta). Note decreased keratin-6 expression and increase of cell cohesion in KN93-treated CerS4^epi−/− organoids (scale bar 20 μm; n = 4 organoids/genotype; mean ± SD; unpaired t test). (F) Representative images of CerS4^epi−/− cells transfected with CerS4-HALO. Note localization in the endoplasmic reticulum. Scale bar 10 µm. (G) Normalized intensity tracks and quantification of single Ctr, CerS4^epi−/−, and CerS4^epi−/− + CerS4-HALO keratinocytes imaged with Fluo-4 AM Ca²⁺ probe to identify oscillations. Grey straight line marks addition of 5 μM KN93. Note that enhanced oscillations were restored to control levels by rescuing CerS4 expression (n = 3 independent experiments with >30 cells/condition/experiment; mean ± SD; Kruskal–Wallis). PCP/CE = planar cell polarity/convergent extension.